Progress 03/01/17 to 02/28/18

Outputs
Target Audience:The target audience for our efforts includes scientists, policy administrators, agriculturalists, households with backyard citrus fruit, members of the citrus industry and the general public. To disseminate information to our target audience, we used diverse platforms to meet all recipients.We established a Website to provide information on citrus and RNAi, along with other technologies that is available to the general public (http://ucanr.edu/sites/scienceforcitrushealth). We also presented posters and talks at various scientific meetings (see below). We participated in the CDRE Project Directors meeting held in conjunction with the Citrus Disease Subcommittee meeting last February 2017, in San Antonio, TX. This group included USDA policy administrators as well as representatives from the California, Florida and Texas citrus industries. We participated in local grower meetings in California and Florida. We presented new scientific information at national and international scientific meetings. Changes/Problems:No problems have arisen, and we anticipate none for the remainder of our effort. What opportunities for training and professional development has the project provided?Undergraduate students, graduate students and postdoctoral scientists have been actively involved in this effort. All have opportunities to interact with other scientists as part of this effort. In addition, several had opportunities to attend local and/or national scientific meetings to present information, meet other scientists and learn new information which will be improtant to our overall research effort. How have the results been disseminated to communities of interest?In addition to publications, websites and blogs resulting from our effort, all PIs have attended various local meetings and presented information to agriculture groups and stakeholders. We had a group advisor meeting for our project in Lake Alfred, FL in November 2017 to get feedback and suggestions for our effort, and presented our project and results at the California Citrus Research Board meeting in Visalia, CA in October 2017. What do you plan to do during the next reporting period to accomplish the goals?We are on track to continue to make excellent progress. Our goals remain the same and we anticipate excellent success from this project.

Impacts
What was accomplished under these goals? Objective 1. Dawson lab -Evaluating approximately 20 RNAi constructs in the CTV vector. Foreign sequences can be expressed from several locations within the CTV genome, allowing modulation of levels of foreign gene expression. RNAi inducer sequences (now 200-300b) that are effective against psyllids are being reconstructed in the CTV vector into smaller segments (50, 100, and 150 nts) and compared to the activity of the original sequence. We also are evaluating the optimal position within the CTV vector for expressing RNAi sequences. We have found that some sequences inserted at the 3' end of the vector are less stable. Those sequences are now being tested inserted between p13 and p20 genes. Killiny lab Liquid chromatography-tandem mass spectrometry identified 89 protein spots. Nineteen protein spots were found to be implicated in stress/defense/immunity; 7 in development regulation; 9 in nervous system functions; 4 in the reproductive system; 23 in cytoskeleton and muscle organization; and 4 in movement, flight and other processes. We are evaluating RNAi inducer effects on the non-target natural enemies already used for D. citri biological control, including the two parasitic waspsTamarixia radiata Waterston (Hymenoptera: Eulophidae) and Diaphorencyrtus aligarhensis (Hymenoptera: Encyrtidae), and the beneficial insect honeybee, Apis mellifera. Objective 2. Ng lab We have succeeded in constructing pCAM-T36-CA, a pCAMBIA-based prototype full-length cDNA clone of T36-CA (confirmed by nucleotide sequencing). We have also transformed Agrobacterium tumefaciens strain GV3101 with pCAM-T36-CA and inoculated the resulting transformant to Nicotiana benthamiana plants to initiate virus infection. Results of RT-PCR analyses and immunological assays using systemic leaves sampled from the agroinoculated plants have shown that pCAM-T36-CA is biologically active. Objective 3. Falk lab We have discovered six new viruses of D. citri, and we have 4 of these in culture at UC Davis. In order to maintain these viruses we have also established colonies of D. citri collected from Hawaii, California and Taiwan. We are fortunate that we are able to maintain all D. citri and the D. citri viruses in the UC Davis BSL 3P Contained Research Facility under conditions that ensure their containment. Our primary objective is to engineer at least one of these viruses and then to use it to induce desirable traits in D. citri. Such traits could be interfering with the ability of D. citri to transmit CLas, reduced D. citri fecundity or lifespan. We have generated full-length cloned cDNAs for three of these viruses, including DcDNV, DcACV and Diaphorina citri picorna-like virus (DcPLV). We do not yet have DcPLV in culture, it has been found only in Brazilian D. ctiri and we cloned it from D. citri collected in Brazil. We have recently obtained a USDA APHIS permit to import D. citri from Brazil Objective 4. Keesling The Keesling lab has developed a mathematical model of CLas spread based on the lifecycle of D. citri populations. The model takes into account factors such as psyllid movement patterns, psyllid aging and mortality, and citrus flushing patterns. This model is highly adaptable and is capable of incorporating factors such as pesticide spraying, invasion of psyllids from other groves, and the various effects that RNAi constructs may have on the psyllid population or CLas transmission. Although we currently model the spread of CLas, we aim to develop a further model that predicts the development of HLB symptoms after infection by CLas. After the model has been sufficiently validated, we will run simulations with a variety of RNAi constructs and other parameters discussed previously. Those with sufficiently powerful effects will be considered for field-testing. We have madeimprovements to our model, including bettermodeling of the role of temperature on flush and psyllid developmentand a more sophisticated treatment of symptom progression. We have recently been provided withdata on the impact of temperature on bacterial titers and transmission that will also be incorporated into the model. Objective 5. Jetter labFLORIDA Several meetings were held with researchers, growers and citrus industry personnel to determine how growers in Florida were currently managing ACP and HLB. The purpose of the trip was to meet with collaborators, learn of their research and meet with Florida growers to determine their interests and what they are doing now. The information would be used to determine the baseline scenarios needed estimate the benefits of the development of the RNAi and Bt-toxin ACP management strategies. CALIFORNIA In California HLB is being found in new regions located in urban areas in the greater Los Angeles Basin, and Riverside County. There is no confirmed HLB yet in commercial acreage; however, a commercial orchard is now in a ACP/HLB quarantine zone following the latest find of HLB in Riverside County.Surveys with growers are being developed to determine the probabilities that they will opt into the recommendations to remove infected trees, opt out, or if there is a financial incentive that will motivate those reluctant to remove trees to opt into the tree removal program. Objective 6. Lemaux, Grafton-Cardwell Our outreach and extension efforts provide information to the citrus industry, citrus growers and the general public. The web site, "Science for Citrus Health" (http://ucanr.edu/sites/scienceforcitrushealth/) was developed as a University of California Ag and Natural Resources (ANR) website, going live in May 2015. Since then it has had ~2500 visits and over 150 downloads from the site (12/7/2017). The website covers the ACP/HLB situation and provides resources for growers to better understand techniques being developed to battle the disease. In addition to updating general information on the website during this period, we worked with citrus project researchers to develop descriptions and images of research strategies and accomplishments and translate them into language understandable by the general public. We termed these pieces, Research Snapshots (http://ucanr.edu/sites/scienceforcitrushealth/Research_Snapshots/ ). The first Snapshot section, Early Detection Techniques (5 entries), has articles on using metabolites, volatile organic compounds, microbial communities, antibodies or starch sensors to detect HLB and the metabolic changes that occur following infection. In a second section, Established Orchards (5 entries), a variety of approaches that are being developed are described. These include using insect viruses to combat Asian Citrus Psyllid or to provide insecticidal or anti-microbial protection, blocking transmission of CLas using Bt toxin to suppress Asian Citrus Psyllid and protecting established orchards using RNAi. In a third section, Replants (5 entries), approaches are described that require replanting of orchards. These include a Bt approach to suppress the Asian Citrus Psyllid, using genome editing to develop HLB-resistant citrus, introducing plant defense genes, and developing founder lines to insert future genetic strategies. In a fourth section, Psyllid (3 entries), approaches aimed directly at the psyllid (developed under the NuPsyllid project) are covered, like efforts aimed at altering the psyllid's beneficial bacteria, using insect viruses to thwart the psyllid's proliferation and stopping spread of CLas using beneficial bacteria. In a new section, Existing Tools (1 entry), efforts are described using existing methods to address HLB, like use of reflective mulches to repel insects.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: El Desouky, A., Ramos, J.E., Hall, D.G., Dawson, W.O., and Shatters, R.G. 2016. Acquisition, replication and inoculation of Candidatus Liberibacter asiaticus following various acquisition periods on huanglongbing-infected citrus by nymphs and adults of the asian citrus psyllid. PLoS ONE doi: 10.1371/journal.pone.0159594.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Galdeano, D. M., Breton, M. C., Lopes, J. R. S., Falk, B. W., Machcado, M. A. 2017. Oral delivery of double-stranded RNAs induces mortality in nymphs and adults of the Asian citrus psyllid, Diaphorina citri. PLoS One 12(3): e0171847.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Killiny, N. and Kishk, A. (2017) Delivery of dsRNA through topical feeding for RNA interference in the citrus sap piercing-sucking hemipteran, Diaphorina citri. Insect Biochemistry and Physiology. 95, e21394.

Progress 03/01/16 to 02/28/17

Outputs
Target Audience:The target audience for our efforts includes scientists, policy administrators, agriculturalists, households with backyard citrus fruit, members of the citrus industry and the general public. To disseminate information to our target audience, we used diverse platforms to meet all recipients. We published five scientific papers in good journals this past year to target scientists. We also published one article in Citrograph, a general citrus information magazine, as a means to get out information to citrus industry members and the public. We established a Website to provide information on citrus and RNAi, along with other technologies that is available to the general public (http://ucanr.edu/sites/scienceforcitrushealth). We also presented posters and talks at various scientific meetings (see below). We participated in the CDRE Project Directors meeting held in conjunction with the Citrus Disease Subcommittee meeting last February in San Antonio, TX. This group included USDA policy administrators as well as representatives from the California, Florida and Texas citrus industries. We presented an oral report and a poster of our work Our outreach and extension effort is designed to provide information to the citrus industry and to the general public in California and Florida. These audiences will be educated utilizing meeting presentations, Fact Sheets, a web site (http://ucanr.edu/sites/scienceforcitrushealth)and powerpoint slides. Changes/Problems:We do not have any major changes. We believe we are on track to meet our objectives as described. What opportunities for training and professional development has the project provided?Undergraduate, graduate and postdoctoral students have been actively involved in the research effort. How have the results been disseminated to communities of interest?See previous sections. We have generated several scientific publicaitons, a website and given a number of talks in different venues. What do you plan to do during the next reporting period to accomplish the goals?We are on track to meet our objectives.

Impacts
What was accomplished under these goals? Objective 1. The Dawson Lab is using the Citrus tristeza virus (CTV) vector to to prevent reproduction of psyllids in citrus seedlings. Although the RNAi expression prevents psyllid development in most trees, it fails in some. The failures appear to be partially due to the combat between the plant expressing RNAi defenses to exclude CTV, and the CTV expressing RNAi suppressors to reduce the RNAi effect. We are down-regulating the CTV suppressors. The Killiny lab is screening interfering RNAs for potential off-target effects in beneficial insects, beginning with honeybees, by injecting dsRNAs directly to the insect and determining any detrimental effects. The Killiny lab also focused on genes implicated on pesticide resistance showing that targeting certain carboxyesterase genes and two glutathione-S-transferase genes increases mortality and susceptibility to pesticides in D. citri. The Florida Citrus Research and Development Foundation (CRDF) is funding Southern Gardens Citrus to conduct a field test of CTV-expressed RNAi sequences to reduce psyllid reproduction and thus spread of HLB. The field test application has been submitted and was approved by EPA and USDA February 24, 2017, with a target date of April 2017 to establish the trial. Objective 2: Cloning of RB25-CA: We have cloned three overlapping cDNA fragments (amplicons) corresponding to the full-length genome of this CTV strain from California. Sequencing the cDNA clones revealed that it differs from that of a fully-sequenced strain of RB25 from Puerto Rico by about 1.7% (or 351 out of 19257 nucleotides). Mistakes observed in the cloned sequences of RB25-CA, including nucleotide insertions, replacements and deletions, have given rise to multiple frameshifts and stop codons that must be fixed. Some of these mutations can be fixed independently i.e. prior to assembly into the pCAMBIA 1380 binary plasmid (see below), while others will be fixed during or after they have been moved into that plasmid depending on the cloning strategy (see below). Our strategy is to systematically replace all the nucleotides of the Florida strain of T36 (in the C86 construct i.e. a pCAMBIA 1380-based construct of the Florida strain of T36) with those of T30-CA or T36-CA. We have also cloned parts of the T30-CA or T36-CA genomes directly into pCAMBIA 1380. We have encountered toxicity issues with some of the products. For example, although we successfully ligated a fixed (mistakes corrected) 11 kb region of the T30-CA with an intermediate construct that contained a small part of the 3' end of C86, this ligated product was toxic to E. coli cells upon transformation. Objective 3. We have lyophilized, frozen, and ethanol preserved D. citri containing several viruses including Diaphorina citri picorna-like virus (DcPLV), Diaphorina citri-associated C virus (DcACV), Diaphorina citri flavi-like virus (DcFLV) and Diaphorina citri densovirus (DcDV). Our effort is primarily focused on DcACV and DcDV. DcACV has a small bipartite positive- sense ssRNA genome, and has some similarities to nodaviruses (e.g. Flock house virus; (FHV)). We have cloned the complete genome of DcACV under the control of an inducible promoter (MT) and are attempting to infect lepidopteran, hemipteran and dipteran cells, with both cDNA clones and in vitro transcription products. We have had very good success with FHV, a well characterized ssRNA nodavirus with a genome organization similar to that of DcACV as a positive control in both S2 and sf9 cells. However, none of these cell lines transfected with DcACV cDNA clones showed consistent replication of DcACV so far (qPCR results show some possible replication compared to the reference gene in S2 cells but the titer of the virus is very low). Our focus now is on psyllid intra-hemocoel micro-injection methods for both FHV and DcACV. Our mortality rate so far is high; however, we have had some progress to optimizing the system by applying different syringes and needles to minimize mortality. In artificial diet feeding tests we have evidence suggesting that we can orally transmit DcDV to naïve D. citri and that viral sequences are retained in some proportion of at least one subsequent D. citri generation. We have also cloned the entire DcDV genome in three fragments and successfully ligated these fragments in vitro to produce a full-length DcDNV clone which can be amplified by PCR. As described for DcPLV, this PCR product will serve as inoculum for transfection into our various cell lines as well as for microinjection into D. citri insects. Objective 4. The Keesling lab continues to improve the mathematical models of HLB spread. They have developed models to predict the effect of RNAi on HLB spread that were the basis of the design of the field test. Objective 5. Developed the baseline management scenarios for Florida and California that will be used in the analysis. Both Florida and Texas will be treated as one region. California will be treated as three regions: San Joaquin Valley, Coast, and Inland Southern California. Developed the market model to estimate the net costs and benefits to consumers and producers of a shift in the supply curve due to changes in the costs of production and lost production. The model can also simulate the effects of changes in the demand for fresh fruit and juice due to different assumptions on the acceptance by consumers of the RNAi technology. Completed the baseline data collection for all baseline market variables needed for each region in the model (production, consumption, trade and market prices) for the U.S. orange, grapefruit and lemon industries. Began programming the economic modeling in Matlab that will be combined with the spread modeling being completed by Keesling (Objective 4). Objective 6. Our outreach and extension effort is designed to provide information to the citrus industry and the general public. We have been working with project researchers to gather descriptions of research strategies and then translating that material into language that can be understood by the general public. In 2015-16 we completed a 'fact sheet', "What Makes Lemons, Oranges and Limes Look and Taste Different?", which contains information on the genetics and genetic engineering as it relates to citrus and psyllids. It also includes information on nongenetic approaches being pursued to combat the HLB problem. This fact sheet will be used as a handout when extension education is conducted on this subject. The web site, "Science for Citrus Health" has been developed as a UC ANR web site, http://ucanr.edu/sites/scienceforcitrushealth/. The web site describes the ACP/HLB situation and provides resources for growers to better understand the techniques that are being developed to battle the disease. Peggy Lemaux, Beth Grafton-Cardwell and Lukasz Stelinski are collaborating on creating descriptions of research projects and approaches that are currently being pursued around the nation to manage HLB (both engineered and non-engineered approaches). Topics include Early Detection Techniques such as Volatile organic compounds and starch sensors for detecting HLB, methods for protecting established orchards such as RNAi for disrupting psyllid transmission and utilizing citrus tristeza virus as a carrier for antimicrobial peptides or insecticides, methods that would create a nupsyllid to release to reduce HLB spread by wild psyllids, and methods that would transform citrus and require replanting of citrus. An extensive collection of PowerPoint slides, covering a diverse array of topics, is posted on the web site. The topics include descriptions of genetic engineering of plants and insects and how these might be used to address HLB, nonengineering approaches, regulatory issues, consumer attitudes, labeling efforts and trade issues. The powerpoint is provided for extension and research personnel to educate growers.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Nigg, J. C., Nouri, S., and Falk, B. W. Complete genome sequence of a putative densovirus of the Asian citrus psyllid, Diaphorina citri. Genome Announc. 4(4):e00589-16. Doi:10.1128/genomeA.00589-16.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Matsumura, E. E., Nerva, L., Nigg, J. C., Falk, B. W., and Nouri, S. Complete genome sequence of the largest known Flavi-like virus, Diaphorina citri flavi-like virus, a novel virus of the Asian citrus psyllid, Diaphorina citri. Genome Announc. 4 doi:10.1128/genomeA.00946-16.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Nouri, S., Salem, N., Falk, B. W. Complete genome sequence of Diaphorina citri-associated C virus, a novel putative RNA virus of the Asian citrus psyllid, Diaphorina citri. Genome Announc 4(4):e00639-16. Doi:10.1128/genomeA.00639-16.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Kishk, A., Anber, H.A., AbdEl-Raof, T.K., El-Sherbeni, A.D., Hamed, S., Gowda, S., Killiny, N. (2017) RNA interference of carboxyesterases causes nymph mortality in the Asian citrus psyllid, Diaphorina citri. Archives of Insect Biochemistry and Physiology. 94, e21377
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Killiny, N., Hijaz, F., Harper, S. J., and Dawson, W. O. (2017). Effects of Citrus tristeza closterovirus infection on phloem sap and released volatile organic compounds in Citrus macrophylla. Physiological and Molecular Plant Pathology. Early view
• Type: Websites Status: Published Year Published: 2016 Citation: http://ucanr.edu/sites/scienceforcitrushealth
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Yu, X., Gowda, S., Killiny, N. 2017. Double-stranded RNA delivery through soaking mediates silencind of the muscle protein 20 and increases mortality to the Asian citrus psyllid, Diaphorina citri. Pest Management Science. DOI: 10.1002/ps.4549

Progress 03/01/15 to 02/29/16

Outputs
Target Audience:The target audience for our efforts includes scientists, policy administrators, agriculturalists, households with backyard citrus fruit, members of the citrus industry and the general public. To disseminate information to our target audience, we used diverse platforms to meet all recipients. We published two papers in top journals this past year to target scientists, and two articles in Citrograph, a trade journal. One of our scientific publications (Nouri et al., 2016) reports our Diaphorina citri virus discovery and characterization. This information is important for other scientists working with D. citri and other insect viruses. In the second publication by Lee et al., 2015, information is reported on modeling HLB spread. This is critical for scientists but also for translational applications of our ongoing efforts. Already in the first year, the Florida Research and Development Foundation has decided to move ahead to establish field tests based on our preliminary results from Objective 1 and according to models develop by the Keesling group (Objective 4; thus targeting the Florida citrus industry). We also presented posters and talks at various scientific meetings (see below). We participated in the CDRE PD meeting held in conjunction with the Citrus Disease Subcommittee meeting. This group included USDA policy administrators as well as representatives from the California, Florida and Texas citrus industries. We presented an oral report and a poster of our work. We also published two articles in Citrograph (Ng et al., 2015; 7(1) 76 - 80; Teiken, C., P. et. al., 2015. 6(1):24-31), a general citrus information magazine, as a means to get out information to citrus industry members and the public. Our outreach and extension effort is designed to provide information to the citrus industry and to the general public in California and Florida. These audiences will be educated utilizing meeting presentations, Fact Sheets, a web site and powerpoint slides. . We have been working with project researchers to gather current scientific information that is being used to create various extension and outreach materials. This involves gathering the information and then translating that material into language that can be understood by those outside science. In this regard we have completed a 'fact sheet', "What Makes Lemons, Oranges and Limes Look and Taste Different?", which contains information on the genetics and genetic engineering as it relates to citrus and psyllids. It also includes information on approaches being pursued to combat the HLB problem. Given that it will be available as an online resource, it can be constantly updated with emerging information. This fact sheet will be used as a handout when extension education is conducted on this subject. The web site, "Science for Citrus Health" is under development and will be introduced in the coming months.On this site, among other general information, we provide simple descriptions of research projects and approaches that are currently being pursued to manage HLB (both engineered and non-engineered approaches) for both agriculturalists and homeowners. An extensive collection of PowerPoint slides, covering a diverse array of topics, is nearing completion. The topics include descriptions of genetic engineering of plants and insects and how these might be used to address HLB, nonengineering approaches, regulatory issues, consumer attitudes, labeling efforts and trade issues. These slides will be made available on the project website for general outreach use. Changes/Problems:Michael Rogers, one of our original co-PIs is now the Director of CREC, Lake Alfred. We will replace his position on our effort as soon as his replacement is hired. What opportunities for training and professional development has the project provided?Our project has provided very good opportunities for training and professional development for undergraduate and graduate students, and postdoctoral scientists. At UC Davis we had one undergraduate student (Falk lab), two graduate students (1 each, Jetter and Falk labs), 1 visiting graduate student (Falk lab), and two postdocs (Falk lab). At the University of Florida there were three postdocs (2 in Keesling lab, 1 in Killiny lab); and at UC Riverside there was one postdoc and 1 Assistant specialist. How have the results been disseminated to communities of interest?Publications: Ng, J.C.K., Chen, A.Y.S., and Yokomi, R. 2015. Fighting HLB with a Citrus tristeza virus (CTV)-based vector-heterogeneity in the genome ends of CTV is an important consideration. Citrograph 7(1), 76-80. Lee, J.A., Halbert, S.E., Dawson, W.O., Robertson, C., Keesling, J.E., and Singer, B.H. 2015. Asymptomatic spread of huanglongbing and implications for disease control. Proc. Natl. Acad. Sci. USA 112: 7605-7610. (doi/10.1073/pnas.1508253112) Nouri, S., Salem, N., Nigg, J. C., Falk, B. W. 2016. Diverse array of new viral sequences identified in worldwide populations of the Asian citrus psyllid (Diaphorina citri) using viral metagenomics. J. Virology 90: 2434 - 2445 Teiken, C., P. Lemaux, B. Grafton-Cardwell and N. McRoberts. 2015. Genetic engineering to protect citrus from HLB. Citrograph 6(1):24-31. Meeting presentations and posters: Chen A.Y.S., Watanabe, S., Yokomi, R., and Ng, J.C.K. Nucleotide heterogeneity at the genomic 5'- and 3'-termini of California (CA) isolates of Citrus tristeza virus (CTV) (Poster presentation, Aug 3, 2015; Annual APS meeting 2015, Aug 1-5; Pasadena, California. Nouri, S., Salem, N., Nigg, J., and Falk, B. W. Diaphorina citr picorna-like virus: A new type of insect virus discovered in the Asian citrus psyllid. Oral presentation at the 34th Annual Meeting of the American Society for Virology. London, Canada. Nouri, S., Salem, N., Falk, B. W. Next generation sequencing for identifying known and novel viruses in the Asian citrus psyllid, the vector of C. Liberibacter asiaticus. (Poster presentation, Aug 3, 2015; Annual APS meeting 2015, Aug 1-5; Pasadena, California. Falk, B. W., Dawson, W. O., Grafton-Cardwell, E., Jetter, K. M., Keesling, J., Killiny, N., Lemaux, P., Ng, J., Rogers, M., Shatters. R. Non-transgenic, near-term RNA interference-based application strategies for managing Diaphorina citri and citrus greening/Huanglongbing (HLB). Poster and oral presentations at the CDS and CDRE program, and USDA Stakeholders meeting, Riverside, CA, 2/16/2016. What do you plan to do during the next reporting period to accomplish the goals?We are on track for objectives 1, 2 and 3, these efforts will continue as described. Co-PIs in both objectives 4 and 5 are working have begun working together and this will enhance our overall efforts. The group for objective 6 also is on track. Specific examples for the next reporting period include: Objective 4. Perfecting the model. The model effectively predicts the asymptomatic spread ofHLB. We are adding a component to the model to predict thetime to symptoms. We have made significant progress andare working to validate the expanded model. Training and professional development. We will be including some graduate students in the project, atleast two this next year. Disseminating results. We will arrange to make presentations at meetings of growers and professionals and publish our results. Design of testing effectiveness. Our model has been used to aid in the design of the plannedfield tests to measure the effectiveness of the CTV vector withits sequences targeting psyllids. We will continue this effort aswork on the field tests continue. The outreach group (Objective 6) will be completing the web site Science for Citrus Health and the powerpoint for training growers and homeowners about genetic engineered and non-engineered strategies for managing the ACP/HLB situation. As components of the research in this project come to fruition, the outreach group will organize grower meetings to discuss the results and update the web, ppt and fact sheets to support the meetings. Objective 5. The goal for the upcoming project year is to develop a bio-economic model for both Florida and California that can be used to estimate the expected costs of ACP/HLB spread if no new technologies are developed. This model will serve as the baseline scenario and can be used to 1) determine the least cost solution to the citrus industries given the current pest and disease status in each region and 2) estimate the benefits to producers and consumers of using genetic engineering to treat ACP or HLB in future years. Changes Problems: Michael Rogers, one of our original co-PIs is now the Director of CREC, Lake Alfred. We will replace his position on our effort as soon as his replacement is hired.

Impacts
What was accomplished under these goals? Objective 1.The Shatters lab is testing different targets of psyllids by feeding double-stranded RNAs. They have identified a series of susceptible targets that when affected result in toxicity to adult psyllids or prevent psyllid reproduction. The Killiny lab is focusing on genes targets implicated in psyllid metamorphosis. They have used proteomics to identify targets for RNAi to disrupt the psyllid development, lifespan, or ability to transmit CLas. These are being tested for efficacy. After effective RNAi inducers are identified by the other labs, the Dawson lab constructs those sequences into the CTV vector and tests their efficacy in citrus against psyllids. They have optimized a 30 day screening procedure that measures the effect of CTV expressed target sequences against psyllid genes for (1) survival of adults on citrus, (2) production of progeny psyllids, (3) acquisition of Clas by progeny, and (4) infection of trees. Objective 2. We purified double stranded (ds)RNA from citrus plants infected with each of two California genotypes of CTV, T36-CA and T30-CA. Sequence analyses of multiple clones showed that the 5' end terminal nt sequences of T36-CA were similar to that of a T36 genotype from Florida (T36-FL; GenBank AY170468). Two core nt sequences were observed, some with additional nts incorporated upstream of the 5' terminal nt. For T30-CA, a comparison of the cloned sequences revealed that most of the T30-CA sequences had an extra A or a guanylate (G) upstream of the first nt in the (+)-RNA and an extra C downstream of the last nt in the (-)-RNA. Most of the 3' end sequences were conserved, with a small number having one or two heterogeneous nts at the extreme ends. We were able to design specific oligo primers for use with reverse transcription (RT) and long range PCR to generate the cDNAs that correspond to two overlapping fragments of the genomes of these two CTV genotypes and cloned the cDNAs. Objective 3. We used a metagenomic approach to analyze viral sequences associated with different worldwide populations of D. citri. By sequencing small RNAs and the transcriptome coupled with bioinformatics analysis, we found that the virus-like sequences of D. citri are diverse. Different viruses are present in geographically different populations. We identified novel viral sequences belonging to the Picornavirus super family, and the Reoviridae, Parvoviridae and Bunyaviridae families, and an unclassified positive-sense single-stranded RNA virus. Moreover, a Wolbachia prophage-related sequence was identified. Our results provide valuable information on new D. citri viruses. Our ongoing efforts are to assess their potential to be used as biocontrol agents, and to engineer them for RNAi approaches. Objective 4. The Keesling lab developed a model for the asymptomatic spread of CLas, the causal agent of HLB, that demonstrates that the disease is spread much faster than previous thought. During the time that an infected female psyllid lays eggs on new citrus flush, that flush becomes infected with CLas. As the progeny nymphs develop, they acquire CLas and the resulting psyllid adults move to new trees with 20-30 days. The psyllid progeny from infected mothers do not need to find an infected source tree to spread the disease. At the time that the infected psyllids leave the tree, only the limited area of the new flush areas where the psyllids reproduced is infected. Long after the infection front of the disease has moved beyond these trees do they develop symptoms and begin to decline. The Florida Citrus Research and Development Foundation has decided to move forward toward using the CTV vector expressing target sequences against psyllids in the field to protect new plantings of citrus agains HLB. Design of the field tests are based on the modeling by the Keesling laboratory and the identification of the most effective sequences. Since the major impact of the RNAi control of psyllids is to reduce spread the disease by reducing the number of psyllids, an effective field test will measure the reduced rate of spread of HLB. The RNAi procedure prevents production of psyllids within the plot but does not prevent the infection of the original tree. The Keesling models are designed to help design the field test in terms of necessary size and to help obtain understanding and data faster since an effective measure is needed in Florida as soon as possible. Objective 5. A literature review was completed on bioeconomic modeling of invasive species. The literature review indicates roughly three approaches that have been taken. The first two approaches involve dynamic equations, and the third a two-period market modeling approach. The first dynamic modeling approach is to use an optimal control model. These models specify an objective function such as minimizing all costs associated with invasive species and their management given biological information on how the pest spreads in the new environment. The benefit of this approach is that it provides a solution to a system of equations on what is the optimal amount of control of the invasive species. The drawback is that for systems with significant spatial heterogeneity, the model can be computationally difficult to solve. The second dynamic modeling approach is to use simulation models. These models simulate the spread of invasive species given their natural rate of spread in the environment, spread under different assumptions about management and the cost of management. These models are computationally more tractable, however the optimal solution must be determined after multiple simulations over different assumptions on how management options affect spread and costs. The third approach is to use two-period market equilibrium models. These models calculate the net benefits to producers and consumers due to changes in costs to producers due to the presence of ACP and HLB. It computes these values based on a before and after scenarios. They are computationally the simplest to complete, and provide an estimate of costs and benefits to both consumers and producers. The models make assumptions on what will be the most reasonable approach to pest and disease management given current knowledge and recommendations to growers. Objective 6. Our outreach and extension effort is designed to provide information to the citrus industry and the general public. We have been working with project researchers to gather descriptions of research strategies and then translating that material into language that can be understood by the general public. In this regard we have completed a 'fact sheet', "What Makes Lemons, Oranges and Limes Look and Taste Different?", which contains information on the genetics and genetic engineering as it relates to citrus and psyllids. It also includes information on nongenetic approaches being pursued to combat the HLB problem. This fact sheet will be used as a handout when extension education is conducted on this subject. The web site, "Science for Citrus Health" is under development and will be introduced in the coming months. On this site, among other general information, we provide simple descriptions of research projects and approaches that are currently being pursued to manage HLB (both engineered and non-engineered approaches). In 2014-15, we presented a series of lectures and quizzes of citrus growers that resulted in a Citrograph article on grower attitudes towards genetic engineering (Teiken et al. 2015). An extensive collection of PowerPoint slides, covering a diverse array of topics, is nearing completion. The topics include descriptions of genetic engineering of plants and insects and how these might be used to address HLB, nonengineering approaches, regulatory issues, consumer attitudes, labeling efforts and trade issues.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Nouri, S., Salem, N., Nigg, J. C., Falk, B. W. Diverse array of new viral sequences identified in worldwide populations of the Asian citrus psyllid (Diaphorina citri) using viral metagenomics. J. Virology 90: 2434 - 2445.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Lee, J.A., Halbert, S.E., Dawson, W.O., Robertson, C., Keesling, J.E., and Singer, B.H. 2015. Asymptomatic spread of huanglongbing and implications for disease control. Proc. Natl. Acad. Sci. USA 112: 7605-7610. (doi/10.1073/pnas.1508253112)
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Teiken, C., P. Lemaux, B. Grafton-Cardwell and N. McRoberts. 2015. Genetic engineering to protect citrus from HLB. Citrograph 6(1):24-31.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Ng, J.C.K., Chen, A.Y.S., and Yokomi, R. 2015. Fighting HLB with a Citrus tristeza virus (CTV)-based vector-heterogeneity in the genome ends of CTV is an important consideration. Citrograph 7(1), 76-80.